The t(8;21) is one of the most common genetic abnormalities in acute myeloid leukemia, identified in 15% of all cases. This translocation generates the fusion protein, AML1-ETO, which contains the N-terminal region of the AML1 protein, including the runt DNA binding domain, and almost the entire ETO protein at the C terminal region. This proposal tests the hypothesis that the t(8;21) fusion protein AML1-ETO requires additional mutations for leukemogenesis and aims to identify molecular pathways associated with the additional mutation. Study of AML1 knockout mice demonstrates that AML1 plays a critical role during hematopoiesis. Our analysis with AML1-ETO knock-in mice shows that AML1-ETO dominantly blocks AML1 function during early hematopoietic cell commitment. Furthermore, our data suggest that AML1-ETO expression is not sufficient to cause leukemia. The studies proposed in Specific Aim #1 will investigate the effect of AML1-ETO on hematopoietic cell commitment using ES cells with AML1-ETO knocked into the AML1 locus. The studies proposed in Specific Aim #2 will study the involvement of sex chromosomes in the development of t(8;21) associated acute myeloid leukemia using genetically engineered mice. The studies proposed in Specific Aim #3 will investigate the abnormal expression or the mutation of additional genes associated with the development of t(8;21) involved acute myeloid leukemia in transgenic mice. We have established animal models with either inducible or myeloid specific expression of AML1-ETO. The experiments proposed will address fundamental questions about the factors critical for normal blood cell differentiation and how t(8;21) is related to the development of leukemia.